Effect of salinity on the oviposition and growth of Ochlerotatus togoi

Abstract Ochlerotatus togoi is a salt‐tolerant euryhaline mosquito that lays its eggs in rock pools. Although it is a pest that can transmit flaviviruses and filarial worms to humans, ecological studies have not been previously conducted because of its limited habitat. However, rising sea levels have created a more favorable environment for Oc. togoi, increasing the risk of Oc. togoi‐borne diseases. We examined the oviposition and growth rates of Oc. togoi at 0–35 psu to obtain ecological data. It exhibited the highest oviposition preference at 0 psu; however, the hatching rate was highest at 10 psu, the pupation rate was highest at 25 psu, and the emergence rate was highest at 5 psu. Oc. togoi showed the highest rate of growth into adults at 25 psu. The results were assessed using Mann–Whitney U and Kruskal–Wallis H tests (post hoc test: Bonferroni), and a regression equation was generated for the incidence of adult Oc. togoi based on the change in salinity (y = −14.318 + 9.821x; y = adult incidence rate; x = salinity). The oviposition habits and developmental conditions of Oc. togoi were confirmed, and the incidence of Oc. togoi based on changes in sea level and ocean salinity was predicted. The results of this study will be useful for controlling salt‐tolerant vectors and responding to vector‐borne diseases.

Approximately 95% of mosquitoes worldwide live only in fresh water; however, Oc. togoi is a salt-tolerant euryhaline mosquito that has a wide habitat ranging from fresh water to salt water (Clark et al., 2004;Jude et al., 2012;Sweeney, 1987;White et al., 2013).
It primarily lives along the coast, and is usually found in rock pools containing a mixture of seawater and rainwater (Hong et al., 1991;Lee & Hwang, 2018;Seo & Chung, 2019).In high-salt environments, larvae inevitably have a high concentration of ions such as Na + , K + , Cl − , and PO 4 3− flowing into their bodies while feeding.
The specific physiological mechanisms of Oc. togoi that control salt concentrations are unknown.It may be that this species has welldeveloped osmotic organs, including the gastric caeca, Malpighian tubule, midgut, hindgut, rectum, and anal papilla, can maintain a balance of ion concentrations even in high-salt environments (Bradley et al., 1984).Osmotic control mechanisms maintain balanced ion concentrations in the hemolymph by actively excreting salt ions.In addition, a large number of mitochondria are present in the epithelial cells of the anus, which may be involved in active ion excretion (Asakura, 1980).
According to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change (IPCC, August 2021), average global temperatures in 2011-2021 were increased by 1.09°C compared with 1850-1900 (Pörtner et al., 2022).Thermal expansion of oceans and melting of glaciers and permafrost as a result of increased global temperatures have contributed to a rise in sea levels (Church et al., 1991;McKay et al., 2011;Wigley & Raper, 1987).The average rate of sea level rise was 1.3 mm/year from 1901 to 1971, but increased by approximately 2.85-fold to 3.7 mm/year from 2006 to 2018, and the rate is expected to continue increasing.When sea levels rise, seawater enters groundwater and rivers, causing coastal lowlands, wetlands, and lagoons to rise and overflow (Cho et al., 2004;Cho & Maeng, 2007;Kim & Lee, 2010;Nicholls et al., 2007).Thus, an increase in salinity in the freshwater environment close to the sea leads to the creation of environments that are more favorable to salt-tolerant mosquitoes, such as rock pools.Salt-tolerant mosquito-borne diseases may increase because of this increase in the population of salttolerant mosquitoes (Ramasamy & Surendran, 2012).In fact, there were cases in 1943 and 2004 where the population of Anopheles labranchiae and Anopheles sundaicus, salt-tolerant mosquitoes that transmit malaria, increased due to an increase in the saline environment.At this time, the incidence of malaria was increased (Geissler & Guillemin, 2010;Krishnamoorthy et al., 2005;Sinka et al., 2011).Likewise, Oc. togoi have the potential to increase the incidence of diseases they mediate due to changes in their environment.Therefore, ecological studies are required to control Oc.
togoi, which is potentially dangerous.In this study, we hypothesized that the oviposition preferences and growth rates of Oc.
togoi would vary with salinity.We collected ecological data by conducting experiments on the oviposition, growth rate, and incidence of Oc. togoi based on changes in salinity.

| Collection and classification
Samples of Oc. togoi were collected from Geoje, Jeju, and Pohang, South Korea.Larvae were collected from stagnant water in artificial structures or rock pools near the coast (Table 1).The collected samples were classified according to the salinity of the habitat (Geoje: 0.5, 0.45, 50 psu; Jeju: 1, 12, 35 psu; Pohang: 1.2, 24, 45 psu), and then transferred to the insectary (temperature 27°C ± 1°C, humidity 70%, L:D = 12:12) of the Animal Systematics & Taxonomy Laboratory of Kyungpook National University.They were raised under the same salinity in the insectary as that found at the collection points.The hatched adults were then used for the oviposition preference experiment, and the eggs laid by the adults were used for growth rate experiments.The collected Oc. togoi were morphologically classified according to taxonomic keys described previously (Lee, 1987;Ree, 2003).Molecular identification (forward primer: 5′-AGG ACA CAT GAA CAC CCA CA-3′; reverse primer: 5′-AGG CGG TGG AGT GTA TGG-3′) was performed as described previously (Bang et al., 2021).

| Oviposition preference
Adult Oc. togoi females (n = 100) collected from the same collection points were randomly selected and set as one group.The experiment comprised nine groups based on the three collection sites (Table 1).The Oc. togoi was placed into an adult mosquito cage containing 10% sugar cotton and fed for 3 days.An acrylic case contained eight salinity groups (0, 5, 10, 15, 20, 25, 30, and 35 practical salinity units [psu]) created by combining aquarium salt with distilled water.The number of eggs laid over 24 h at each salinity was counted (Figure 2).Each concentration was replicated three times, yielding

| Growth rate
As with oviposition preference, the experiments were conducted in an acrylic case divided into eight salinity groups.The growth of Oc. togoi was determined by placing 100 eggs into each section.All eggs were oviposited under the same conditions in the insectary (temperature 27°C ± 1°C, humidity 70%, L:D = 12:12) and all developing larvae were provided the same amount of food.The hatching rate was determined using the cumulative number of larvae hatched from the eggs, the pupation rate was determined using the cumulative number of pupae from the larvae, and the emergence rate was assessed using the cumulative number of adults emerging from the pupae.Finally, the adult rate (the number of eggs that made it to the adult stage) was determined using the cumulative population of adults grown from eggs.A total of nine growth rate experiments were conducted for Oc.
togoi collected from Geoje, Jeju, and Pohang, in triplicate.Oc. togoi was collected from areas with salinities that were as similar as possible to minimize variables due to differences in salinity at the collection point (Geoje: 0.5 psu; Jeju: 1 psu; Pohang: 1.2 psu).

| Ae. albopictus
To compare the salt tolerance of Oc. togoi, a freshwater species, Aedes

| Statistical analysis
Differences in average growth rates between Ae. albopictus and Oc.
togoi were assessed using a Mann-Whitney U test.Using a Kruskal-Wallis H test, the average growth rates of Oc. togoi collected from Geoje, Jeju, and Pohang and Ae.albopictus were compared to establish statistical significance among the four groups.To confirm which of the four groups exhibited statistical significance, a post hoc analysis was performed using Bonferroni's multiple comparison test.

| Oviposition preference
In a total of 27 oviposition preference experiments of Oc. togoi, a total of 20,210 eggs were laid, and the highest oviposition preference was 28.89% (5839 eggs) at 0 psu ( F I G U R E 2 Acrylic case for the oviposition preference experiment of Ae. albopictus and Oc.togoi (7 cm × 7 cm × 7 cm in one compartment).
preference of Oc. togoi decreased as salinity increased; however, there was a large deviation among salinity groups (Figure 3a).In particular, in the ≥15 psu group, the oviposition rate was markedly decreased; the average oviposition preference in the 0-10 psu section was 22.89%, whereas that in the 15-35-psu section was 6.27%, or approximately 3.65-fold lower.
When the oviposition preference of Oc. togoi was evaluated by collection site, the oviposition preference was high for low-salinity environments and decreased significantly after 15 psu (Figure 3a).
The groups with the highest oviposition preference by site were 0 psu (34.39%) at Geoje, 0 psu (29.07%) at Jeju, and 5 psu (18.88%) at Pohang.All three sites exhibited a decreasing tendency as salinity increased.
As the salinity of the oviposition site increased, Ae. albopictus tended to avoid oviposition.Unlike Oc. togoi, Ae. albopictus showed little variation in oviposition preference (Figure 3a).It was highest at 0 psu (40.66%) and rapidly decreased at 10 psu.As with Oc. togoi, no significant difference in oviposition preference was observed between 15 and 35 psu (Table 2).

| Hatching rate
During the hatching rate experiment, 4394 larvae hatched from a total of 7200 Oc. togoi eggs, and the highest hatching rate of all sites was at 10 psu (72.11%) (Table 2).The hatching rate decreased as salinity increased with the lowest rate observed at 25 psu (51.67%) (Figure 3b).The hatching rate of the eight salinity groups was 61.03% on average; those in the 5-20 psu range were higher than average, while hatching rates in the 0 and 25-35 psu ranges were lower than average.Thus, the hatching rate of Oc. togoi showed a decreasing pattern after a peak, which was particularly conspicuous in Oc. togoi collected from Pohang and Jeju (Figure 3b).The average hatching rates at Geoje and Jeju were similar, at 63.38% and 64.29%, respectively, whereas that at Pohang was lower, at 55.42% (Table 2).
Since the results of the Shapiro-Wilk test were non-normal for growth rates at each developmental stage of Oc. togoi and Ae.albopictus, a nonparametric statistical test was performed.The Mann-Whitney U test revealed that the hatching rates were statistically significant for all salinity groups (p < .05;Table 3).In the Kruskal-Wallis H test for regional analysis, salinity groups in the 5-35-psu range showed significant differences (p < .05;Table 4).In the post hoc analysis, no group showed a significant difference at 0 psu, but at 5 psu, the hatching rates of C and J were significantly different (Table 5).The groups that showed significant differences in the 10-35-psu range were C and G, C and J, and C and P (G: Geoje Oc. togoi; J: Jeju Oc. togoi; P: Pohang Oc. togoi; C: Ae. albopictus).

| Pupation rate
For Oc. togoi, a total of 917 of 4394 larvae pupated, and those that pupated at 0 psu had the lowest pupation rate (75; 14.23%) (Table 2).For all Oc.togoi groups, pupation rates peaked at 25 psu (30.32%).The average pupation rate for the entire group was 20.87%, and that in the 0-15 psu range was 16.38%, which was lower than the overall average.The average pupation rate in the 20-35 psu range was 26.08%, which was higher than the overall average.With respect to collection site, the highest pupation rate was found at 25 or 30 psu, followed by a decrease at higher salinities (Figure 3c).The average pupation rates for Geoje and Jeju were 17.16% and 17.50%, respectively, whereas the average pupation rate for Pohang was 29.02%.
For Ae. albopictus, a total of 179 out of 544 larvae pupated, and there were no pupae in salinity groups of ≥15 psu (Table 2).The highest pupation rate was 46.94% at 0 psu, but it rapidly decreased to 26.36% at 5 psu and to 1.79% at 10 psu (Figure 3c).
The Mann-Whitney U test revealed that the pupation rates in the range of 10-35 psu were statistically significant (p < .05;Table 3).In the Kruskal-Wallis H test, pupation rates at 10-35 psu also showed a statistically significant difference (p < .05;Table 4).In the post hoc analysis, no group showed a significant difference between 0 and 5 psu.For the salinity groups in the range of 10-35 psu, comparisons between C and G, C and J, and C and P showed a statistically significant difference (Table 6).
In the case of Ae. albopictus, 175 of 179 pupae developed into adults.The emergence rate was 100% at 0 psu and 93.65% at 5 psu, whereas no emergence was observed above 10 psu (Table 2).
The Mann-Whitney U test revealed that emergence rates were statistically significant over the entire range of 0-35 psu (p < .05; Table 3).The results of the Kruskal-Wallis H test were also statistically significant for the entire range (p < .05;Table 4).The post hoc analysis revealed that difference in the emergence rate between C and G, C and J, and C and P were statistically significant at 0, 10, and 15 psu (Table 7).Statistical significance at 5 psu was observed for differences in emergence rates between J and C and between J and P, whereas statistical significance at 20 psu was observed between C and G and between C and P. At 25 psu, differences in the emergence

| Adult rate
During the entire growth rate experiment, 805 adults emerged from a total of 7200 eggs.As salinity increased, the adult rate increased, showing a peak value of 13.78% at 25 psu and subsequently decreasing (Table 2).The average adult rate for the eight salinity groups was 11.18%, and the 15-30-psu range showed a higher adult rate compared to the average.In other words, the higher the salinity of the larval habitat, the higher the number of Oc. togoi, with a decrease when the salinity exceeded 30 psu.The adult rates in the samples collected from Geoje, Jeju, and Pohang showed similar tendencies, decreasing after a peak (Figure 3).The average adult rate at each site was similar for Geoje (9.63%) and Jeju (9.50%), but for Pohang, the adult rate was higher (14.42%).
For Ae. albopictus, 174 adults emerged from a total of 900 eggs.The adult rate was the highest at 0 psu (23.00%), but at 5 psu, it decreased to 11.80%.No individuals emerged above 10 psu (Table 2).
In the Mann-Whitney U test, adult rates at 0 psu and 10-35 psu showed statistical significance (p < .05,Table 3).In the Kruskal-Wallis H test, adult rates were statistically significant over the entire range of 0-35 psu (Table 4).The post hoc analysis revealed that differences in adult rates between C and G, C and J, and C and P were statistically significant at 0, 15, 20, and 35 psu (Table 8).At 5 psu, the adult rates between C and J and between C and P were statistically significant.At 10 psu, the adult rates between C and G, C and J, C and P, and J and P were statistically significant.At 25 psu, the differences in adult rates between C and G, C and J, C and P, P and G, and P and J were statistically significant.At 30 psu, the differences in adult rates between C and G, C and J, C and P, and P and G were statistically significant.

| Collection site and collection period for Oc. togoi
Oc. togoi larvae were collected from rock pools at three sites with a wide range of salinities (0.5-50 psu; Table 1).Considering that the average salinity of the South Korean ocean is 33 psu, it was confirmed that Oc. togoi grows in habitats with a wide range of salinities, from habitats with relatively low salinity to those with salinity higher than that of the ocean (NIFS, 2022).

| Oviposition preference
The 900 female Oc.togoi used in the oviposition preference experiment showed similar oviposition preferences, regardless of the salinity in which they lived during the larval stage (Table 2; Figure 3).The

TA B L E 8 (Continued)
Moreover, we found that Ae. albopictus did not grow into adults at 10 psu or higher.It appears that salt concentration plays an important role in larval development.In freshwater mosquitoes, organs that control osmosis are less developed compared with those of salt-tolerant mosquitoes.Therefore, it appears that the larvae of Ae.
albopictus died because the equilibrium of ions in the body collapsed as they failed to discharge ions into the hypertonic environment.
However, the shell of the egg serves as a block from the outside.
Furthermore, the hard cuticle layer of the pupa protects the inside from the external environment.Eggs and pupae do not feed, so the balance of ion concentration in the body does not change.Therefore, the difference in the hatching and emergence rates between 0 and 5 psu was not large.

| Climate change and utilization plan
Abnormal climate conditions caused by an increase in global temperatures can lead to an increase in the incidence of disease vectors.
For Aedes aegypti, which mediates various flaviviruses, the incidence is expected to increase by more than 9.5% (Liu-Helmersson et al., 2019).By the end of the 21st century, it may reach 20%-30%.
With the population of Ae. aegypti increasing, there is concern that the incidence of diseases will concomitantly increase.Based on information provided by the IPCC and the Korea Hydrographic and Oceanographic Administration, between 2006 and 2018 the rate of sea level rise increased at an average annual rate of 3.36 mm/year and will continue to increase (KHOA, 2022;Pörtner et al., 2022).In addition, ocean salinity in South Korea is decreasing by 0.006 psu per year (NIFS, 2022).This trend is consistent with the global salinity trend (Rhein et al., 2013).Under these climate change conditions, Oc. togoi is likely to increase.
Interestingly, in 1943, the Pontine Marshes in southern Rome, Italy flooded, which caused an inflow of seawater.During this time, the number of salt-resistant Anopheles labranchiae increased, and as a result, there was a rapid increase in malaria outbreaks (Geissler & Guillemin, 2010).A similar example was found following the 2004 Indian Ocean earthquake, in which salt-tolerant Anopheles sundaicus rapidly increased due to an inflow of seawater to the Andaman and Nicobar Islands of India, resulting in an increased incidence of malaria (Krishnamoorthy et al., 2005;Sinka et al., 2011).
This study provides information on the oviposition and growth of Oc. togoi based on salinity.The findings of this study will serve as basic data for controlling Oc. togoi and responding to mosquitoborne diseases.

| CON CLUS IONS
In this study, Oc. togoi and Ae.albopictus preferred 0 psu at the oviposition site; however, Oc. togoi showed strong tolerance to salinity.
Ae. albopictus adults grew only at 0 and 5 psu, but Oc. togoi adults grew in all salinity groups.The hatching rate of Oc. togoi increased as salinity increased but subsequently decreased after a peak (72.11%) at 10 psu.The pupation rate of Oc. togoi increased as salinity increased, but decreased after a peak (30.32%) at 25 psu.The emergence rates of Oc. togoi were 94.67% at 0 psu and 95.92% at 5 psu.
The emergence rate at salinities of ≤10 psu decreased as salinity increased.Overall, Oc. togoi showed the highest adult rate at 25 psu (13.78%).
albopictus, was used.Ae. albopictus populations were provided by The Korea Disease Control and Prevention Agency.They were raised individually in the insectary of the Animal Systematics & Taxonomy Laboratory of Kyungpook National University.The oviposition preference and growth rate experiments were conducted in the same way as those for Oc.togoi and repeated three or five times, respectively.
between C and G, C and J, C and P, P and G, and P and J were statistically significant.At 30 psu, emergence rates significantly differed between C and G, C and J, C and P, G and P, and G and J.At 35 psu, emergence rates significantly differed between C and G, C and J, C and P, and G and P.
Mann-Whitney U test of Ae. albopictus and Oc.togoi growth rates by salinity.
TA B L E 3 Kruskal-Wallis H test of Ae. albopictus and Oc.togoi growth rates by salinity.Post-hoc analysis of Bonferroni's multiple comparison test on the adult rate by G, J, P and C.
of larvae into adults takes approximately 2 weeks to 3 months.During this period, the salinity of rock pools can change as a result of environmental factors, such as temperature, relative humidity, wind velocity, waves, and sunlight.Therefore, the salinity measured at the time of sample collection was temporary, and it is difficult to correlate salinity with the selection of oviposition sites.TA B L E 4Note: G: Geoje Oc. togoi; J: Jeju Oc. togoi; P: Pohang Oc. togoi; C: Ae. albopictus.TA B L E 5Post-hoc analysis of Bonferroni's multiple comparison test on the hatching rate by G, J, P and C. Note: G: Geoje Oc. togoi, J: Jeju Oc. togoi, P: Pohang Oc. togoi, C: Ae. albopictus.a Statistically significant.TA B L E 5 (Continued) TA B L E 6 Post-hoc analysis of Bonferroni's multiple comparison test on the pupation rate by G, J, P and C. Note: G: Geoje Oc. togoi, J: Jeju Oc. togoi, P: Pohang Oc. togoi, C: Ae. albopictus.a Statistically significant.TA B L E 6 (Continued) TA B L E 7 Post-hoc analysis of Bonferroni's multiple comparison test on the emergence rate by G, J, P and C. a Statistically significant.Note: G: Geoje Oc. togoi, J: Jeju Oc. togoi, P: Pohang Oc. togoi, C: Ae. albopictus.a Statistically significant.